New treatment of chronic ulcers

ABSTRACT

The present invention provides a pharmaceutical formulation comprising LL-37 or a pharmaceutically-acceptable salt thereof and one or more pharmaceutically-acceptable diluent or carrier system, for use in a method of treatment of a chronic ulcer wound (such as a hard-to-heal venous leg ulcer or a diabetic foot ulcer), which method comprises: (a) topical application of said formulation to said ulcer; followed by (b) application of a dressing, and wherein said application of said formulation provides for a dose of LL-37 at the wound site that is below about 80 μg of LL-37 applied per cm2 of wound area, and/or below about 26.7 μg of LL-37 applied per cm 2  of wound area, per day of treatment.

FIELD OF INVENTION

The present invention relates to methods of using wound care products.In particular, the invention provides for the treatment of chroniculcers.

BACKGROUND

Wound healing is a dynamic pathway that optimally leads to restorationof tissue integrity and function. A “chronic” wound is one in which thehealing process is prolonged and incomplete, resulting in a lack ofrestoration of integrity. Chronic wounds are a challenge to the patient,healthcare professionals, and the healthcare system, and significantlyimpair the quality of life for millions of people. Long-term, repetitivetreatment is required, which imparts an enormous burden on society interms of lost productivity and healthcare budgets.

Hard-to-heal (HTH) or chronic ulcers are a class of chronic wound. HTHulcers of the leg and the foot, are often painful, stigmatising and havea negative impact on functional ability.

Venous leg ulcers (VLUs) are chronic skin and subcutaneous ulcerationsthat occur on the lower leg, between the knee and the malleolus. TheSwedish Medical Product Agency defines a VLU as one that is below theknee caused by venous insufficiency, which does not heal within sixweeks of standard therapy (i.e. traditional wound care products, such aslow technology gauze-based dressings, woven and non-woven sponges,conforming bandages and non-adherent bandages).

Chronic venous insufficiency (CVI) results when the veins in the legslose the ability to pump venous blood effectively. A range of problemsresult from leakage of fluid into the interstitial space and the area isthen prone to ulceration.

VLUs constitute the majority of all leg ulcers accounting for 70-90% ofwounds in lower extremities (see Snyder, Clin. Dermatol., 23, 388(2005)). Epidemiological data suggests that 1.5-3.0 people per 1,000(0.15-0.3%) have active leg ulcers, rising to around 20 people per 1,000(2%) in individuals over 80 years of age (see Valencia et al, J. Am.Acad. Dermatol., 44, 401 (2001)). Consequently, VLUs are a massiveeconomic burden. In Europe alone, the total annual cost is estimated at

6.5 billion based on an average cost of

7000 per episode (see Posnett, et al, J. Wound Care, 18, 154 (2009)).Expense is not limited solely to the direct cost of ulcer care, butincludes indirect costs associated with disability and lost days atwork.

Diabetic foot ulcers (DFUs) are one of the major complications inpatients suffering from diabetes mellitus. Diabetes causes peripheralneuropathy with impaired sensory function and an increased risk ofwounding. Circulatory impairment impedes the process of natural skinhealing thereby resulting in HTH wounds. The risk of a patient withdiabetes mellitus developing a foot ulcer is approximately 25% accordingto the International Diabetes Federation (see e.g. Boulton et al,Lancet, 366, 1719 (2005) and Singh et al, JAMA, 293, 217 (2005)). Theannual incidence and prevalence of diabetic foot ulcers in patients withdiabetes are 2-6% and 4-10%, respectively (see also Ramsey et al,Diabetes Care, 22, 382 (1999)). In addition, 10% of diabetic patientshave contributing factors such as peripheral neuropathy and vascularcomplications for developing foot ulcers. DFUs are prone to infectionsleading to significant morbidity, and are associated with 25-90% of allamputations in this patient population (see also Ragnarson-Tennvall etal, Diabetologia, 44, 2077 (2001)).

DFUs also represent a significant economic burden. In Europe, DFUs thatdo not heal within 12 months cost an average of

20,000 per patient (Prompers et al, Diabetologia, 51, 1826 (2008)).

There is no evidence to support that standard dressing materials are ofany significant benefit to treat the aforementioned HTH ulcers, evenwhen used in conjunction with other standard measures (cleansing,debridement, bandaging, etc.). In Europe, Regranex® (becaplermin) gel isindicated for the treatment of lower extremity diabetic neuropathiculcers that extend into the subcutaneous tissue or beyond and have anadequate blood supply. It is used as an adjunct to, and not a substitutefor, good ulcer care practices including initial sharp debridement,pressure relief and infection control. Although Regranex is the onlydrug product approved for treatment of such HTH ulcers, it has nodocumented effect on VLUs.

Thus, there is an urgent need for improved treatments of chronic wounds,and in particular HTH ulcers such as VLUs and DFUs.

LL-37 is derived from the 18-kDa human cathelicidin antimicrobialprotein 18 (hCAP18). It is an important mediator in tissue repair anddefense against infection. Both hCAP18 and LL-37 are present in variousbody fluids, including wound fluid (see, for example, Murakami et al, J.Dent. Res., 81, 845 (2002), Schaller-Bals et al, Am. J. Respir. Crit.Care Med., 165, 992 (2002) and Sorensen et al, J. Biol. Chem., 278,28540 (2003)). Although, in acute wounds, transcription of hCAP18/LL-37is up-regulated within a few hours, and LL-37 is expressed at the woundmargin by basal epidermal keratinocytes, chronic wounds have no LL-37immunoreactivity at the wound edge (see, for example, Dressel et al,Exp. Dermatol., 19, 628 (2010) and Rivas-Santiago et al, J. Derm. Sci.,65, 19(2012)).

In vivo studies with synthetic LL-37 have shown that subcutaneousinjection stimulates angiogenesis (Koczulla et al, J. Clin. Invest.,111, 1665 (2003)), and repeated topical administration in acute woundsattracts inflammatory cells to the surrounding tissue. Antibodiesagainst LL-37 inhibited healing in an ex vivo model of human acutewounds (Heilborn et al, J. Invest. Dermatol., 120, 379 (2003); see alsointernational patent application WO 2004/067025), while topicaltreatment with LL-37 stimulated wound healing in mice treated with acorticosteroid to impair normal healing (Ramos et al, Peptides, 32, 1469(2011)). Administration of LL-37 in acute wounds of diabetic mice withimpaired wound healing has shown beneficial effect in one study(Carretero et al, J. Invest. Dermatol., 128, 223 (2008)), while otherstudies have failed to show an effect (Steinstraesser et al, PLoS ONE,7, e39373 (2012)). EP 1 358 888 discloses that LL-37 is an inducer ofangiogenesis.

International patent application WO 2009/001087 discloses wound careproducts in which LL-37 is impregnated into a solid substrate (adressing) comprising a wound care material. There is no suggestion inthat document that it is necessary to apply a certain dose of LL-37 in aformulation per unit wound area to treat the wound effectively. There iscertainly no suggestion that there may be an upper limit to that dose,let alone what that limit might be.

DISCLOSURE OF THE INVENTION

The inventors have now found, in the clinical setting, that certaindoses of LL-37 can be applied to treat effectively HTH/chronic legulcers. In particular, as described below, the inventors have found,surprisingly, that doses of at least about 80 μg of LL-37 applied percm² of ulcer/wound area do not effectively treat chronic ulcers,including VLUs.

According to the invention therefore, there is provided a method fortopically treating a chronic ulcer, such as a VLU or a DFU, which methodcomprises:

-   -   (a) topical application to said ulcer of a pharmaceutical        formulation comprising LL-37 or a pharmaceutically-acceptable        salt thereof and one or more pharmaceutically-acceptable diluent        or carrier system; followed by    -   (b) application of a dressing,        wherein said application of said formulation provides for a dose        of LL-37 at the wound site that is below about 80 μg of LL-37        applied per cm² of wound area to treat said ulcer.

There is further provided said pharmaceutical formulation for use atsaid dose in said method to treat a chronic ulcer, as well as the use ofLL-37 for the manufacture of a pharmaceutical formulation for use atsaid dose in said method to treat a chronic ulcer.

The amino acid sequence of human LL-37 is shown below in SEQ ID NO:1:

[SEQ ID NO: 1] LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

LL-37 may be employed in the form of a pharmaceutically acceptable baseaddition salt, including those of alkali metal cations (e.g. potassiumand sodium) and alkaline earth metal cations (e.g. calcium andmagnesium), ammonium or water-soluble amine addition salts such asN-methylglucamine-(meglumine), and the lower alkanolammonium and otherbase salts of pharmaceutically acceptable organic amines, among others.LL-37 may be used in the form of an acetate salt.

The method/uses of the invention are particularly useful in thetreatment of chronic ulcers, including HTH ulcers, venous ulcers,diabetic ulcers and pressure ulcers, Ulcers that may be mentioned inparticular include DFUs and, particularly, leg ulcers, such as VLUs.

As described hereinafter, in the specific formulations exemplifiedherein, a concentration of 0.5 mg of LL-37 per mL of the specificvehicle that was employed, when applied every third day to the ulcer,was calculated to provide an efficacious dose of about 13 μg of LL-37per cm² of wound area. A concentration of 1.6 mg/mL gave an efficaciousdose of 40 μg/cm². However, a concentration of 3.2 mg/mL, resulting in80 μg/cm², gave rise to no clinical benefit.

Accordingly, the dose of LL-37 that is applied per cm² of the wound areato treat ulcers may be below a value within the range of about 40 μg andabout 80 μg of LL-37 (in other words, below a value that is betweenabout 40 μg and about 80 μg of LL-37). Suitable upper limits for thedose of LL-37 that is applied to the ulcer may be about 75 μg of LL-37applied per cm² of wound area, such as about 70 μg/cm², for exampleabout 65 μg/cm², or 60 μg/cm², including about 55 μg/cm², such about 50μg/cm² or about 45 μg/cm². Suitable lower limits of LL-37 applied percm² of wound area may be about 1 μg, such as about 3 μg, including about5 μg, such as about 10 μg/cm², for example about 15 μg/cm², or 20μg/cm², including about 25 μg/cm², such about 30 μg/cm² or about 35μg/cm².

The inventors have found that chronic ulcers, such as VLUs and DFUs, maybe effectively treated with doses below about 80 μg of LL-37 applied percm² of wound area when applied every third day, which translates toabout 26.7 μg of LL-37 applied per cm² of wound area for each day oftreatment.

By “pharmaceutical formulation that provides for a dose of LL-37 that isbelow about 26.7 μg per cm² of wound area per day of treatment”, weinclude or mean that the:

(i) formulation is of a nature; and/or(ii) frequency of application of the formulation is such,that the amount of LL-37 that is available at the wound site to treatthe ulcer does not exceed about 26.7 μg per cm² of wound area for eachday of treatment.

To elucidate, in the specific formulations exemplified hereinafter, aconcentration of 0.5 mg of LL-37 per mL of the specific vehicle that wasemployed (i.e. the specific pharmaceutical formulation describedhereinafter), when applied every third day, provided an efficacious doseof about 13 μg of LL-37 per cm² of wound area, corresponds to about 4.3μg of LL-37 per cm² of wound area for each day of treatment. Similarly,a concentration of 1.6 mg/mL gave an efficacious dose of 13.3 μg/cm² foreach day. However, a concentration of 3.2 mg/mL resulted in 26.7 μg/cm²for each day, but gave rise to no clinical benefit.

In this respect, it is anticipated that concentrations that are lowerthan about 0.5 mg of LL-37 per mL of the same, specific vehicleemployed, if applied more frequently (e.g. every day), may give rise tomore LL-37 being available at the wound site to treat the ulcer, and soprovide for a clinical benefit. The same applies to concentrations ofabout 3.2 mg/mL or more of the same, specific vehicle, applied lessfrequently than every three days.

Similarly, if a different formulation (for example a more extendedand/or a slow release formulation) is employed, the amount of LL-37 thatis available at any one time to treat the ulcer will be lower than isthe case in the formulation specifically exemplified hereinafter. Thiswill result in less LL-37 being available at the wound site to treat theulcer at any given time. Accordingly, a higher concentration of LL-37may be used initially in such a formulation to ensure that a similaramount of LL-37 is available at the wound site to treat the ulcer at anygiven time. Such a formulation may also be applied less frequently togive rise to the same effect.

According to a further aspect of the invention, there is provided amethod of treatment of a chronic ulcer, which method comprises:

-   -   (a) topical application to said ulcer of a pharmaceutical        formulation comprising LL-37 and one or more        pharmaceutically-acceptable diluent or carrier system; followed        by    -   (b) application of a dressing,        wherein said application of said formulation provides for a dose        of LL-37 at the wound site that is below about 26.7 μg (for        example a dose that is below a value that is between about 13.3        μg and about 26.7 μg) of LL-37 applied per cm² of wound area for        each day of treatment to treat said ulcer.

The pharmaceutical formulation that may be employed in the method of theinvention can be formulated for direct (as defined herein) topicalapplication to an ulcer. Non-limiting examples of such formulations areliquid or semi-solid formulations, and include any solution, suspension,emulsion, cream, gel or lotion, provided that it is sufficiently viscousenough at room temperature (e.g. between about 20° C. and about 25° C.)and at atmospheric pressure (e.g. about 1 atmosphere) that it is capableof physically remaining, in a coherent state, at the wound sitefollowing its application.

The liquid or semi-solid formulation should be viscous, and thus not becompletely free-flowing, e.g. under the influence of gravity, at roomtemperature and at atmospheric pressure. However, it should also not sobe so viscous at room temperature and at atmospheric pressure that it isan essentially solid state, and thus incapable of being manipulated withnormal and/or reasonable manual pressure, using an appropriate medicalinstrument, to e.g. spread it over the wound. Formulations may also beapplied to wounds by spraying using an appropriate spray (e.g.pump-action or aerosol) device to deliver the defined dose per unitwould area (see, for example, international patent application WO2011/056116).

Thus, suitable viscosities for the formulation will depend upon theexcipients that are employed, but may be in the range of about 1 toabout 100 Pas, such as about 50 Pas, e.g. about 25 Pas, including about15 or about 10 Pas. The aformentioned viscosity values are dynamicviscosities at room temperature (as hereinbefore defined) andatmospheric pressure (as hereinbefore defined), as measured by astandard viscometer or rheometer.

Pharmaceutical formulations that may be used to apply LL-37 to ulcersinclude aqueous solutions. An aqueous solution is a solution havingphysiologically- or pharmaceutically-acceptable properties (in relationto pH, ionic strength, isotonicity, etc). For example, isotonicsolutions comprising water and other biocompatible solvents, aqueoussolutions, such as saline and glucose solutions, and hydrogel-formingmaterials, may be employed. The aqueous solution can be buffered, withfor example acetate buffer.

The pharmaceutical formulation may also comprisepharmaceutically-acceptable excipients, such as preservatives to preventmicrobial growth, antioxidants, tonicity-modifying agents, colouringagents and the like. In aqueous suspensions, the compositions can becombined with suspending and stabilising agents.

In one aspect of the invention, LL-37 is dissolved in an aqueous solvent(e.g. water or acetate buffer) and an appropriate thickening agent addedto create a viscous, spreadable aqueous soft gel, cream or lotionformulation.

When formulated in aqueous solutions, LL-37 may also be formulated alongwith a thickening agent in order to obtain a cream, gel or lotion ofsufficient viscosity as described hereinbefore. Suitable agents,including hydrogel-forming materials, which includes synthetic polymers,such as polyvinylalcohol, polyvinylpyrrolidone, polyacrylic acid,polyethylene glycol, poloxamer block copolymers, and the like;semi-synthetic polymers, such as cellulose ethers, includingcarboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,methylcellulose, hydroxypropylmethylcellulose andethylhydroxyethylcellulose, and the like; natural gums, such as acacia,carragenan, chitosan, pectin, starch, xanthan gum and the like. Suchmaterials that may be mentioned include pharmaceutical gradepolyvinylalcohols (for example with degrees of hydrolysis in the rangeof about 70 and about 99% (e.g. between about 85 and about 89%), andmolecular weights in the range of about 15,000 and about 130,000).

It may be advantageous to use a formulation that is bio- and/ormucoadhesive.

It is key that the skilled reader understands however that, once he orshe is in possession of the relevant information presented herein,regarding the maximum amount of LL-37 that should be applied per unitarea of the wound (including per day of treatment), he or she will beable to ascertain, using routine techniques, for the excipients (i.e.the diluent(s), carrier(s), the thickening agent(s) and the otheringredients) that are employed, the correct concentration of LL-37 touse in that particular formulation to enable the appropriate dose perunit wound area (per day).

For example, the formulation can be tested in a bioassay for LL-37activity and compared with LL-37 in phosphate-buffered saline (PBS). Thebioassay may be an antimicrobial assay or an assay based on a responsein a eukaryotic cell line. The concentration of LL-37 in theinvestigated formulation at which the biological effect is equivalent tothe effect of a particular concentration of LL-37 in PBS (e g 10 μg/ml)can be used to calculate a dosing factor. For example, if aconcentration of about 20 μg/ml of LL-37 in the investigated formulationis required to achieve the same biological effect as about 10 μg/ml ofLL-37 in PBS, a dosing factor of about 2 can be derived which is used todouble the concentration of LL-37 in the formulation and thus deliverthe desired dose of LL-37 per wound area.

As will be appreciated by the skilled person, the above general routinetechnique is not applicable in the event that one or more of theexcipients that is employed interacts with LL-37 to denude it, tosubstantially prevent its release from the formulation to the woundsite, and/or to produce a synergistic effect, so resulting in the doseof LL-37 per unit wound area per day of treatment being outside theranges mentioned hereinbefore. On the other hand, if an excipientenables a sustained release of LL-37 from the formulation, this shouldbe taken into account when calculating the concentration to employ, asdescribed above.

In the method/uses of the invention, a pharmaceutical formulationcomprising LL-37 as described herein is first applied directly (i.e.without an intermediate step) and topically to the ulcer as describedhereinbefore. Subsequently and separately to the application of theformulation, a dressing is separately applied. The subsequent andseparate application of the dressing may, for example, take place noless than about 5 minutes, such as about 10 minutes (to allow binding ofLL-37 to the wound tissue), but may also take place no more than about20, such as about 30 minutes, of the application of the pharmaceuticalformulation comprising LL-37.

Dressing materials that may be employed may be inert (i.e. substantiallynon-toxic) materials suitable for use in wound care and/or which arecapable of aiding (for example, accelerating) the wound healing processand/or to preventing infection of a wound. For example, the dressing maybe capable of enhancing epithelial regeneration and/or healing of woundepithelia and/or wound stroma. In one embodiment, the wound care productmay be capable of enhancing the proliferation of epithelial and/orstromal cells.

The dressing may also be capable of absorbing wound exudate.

The dressing may thus comprise a material selected from the groupconsisting of alginates, sheet hydrogels, hydrofibres, foams andmixtures thereof.

Additional dressings, which are capable of absorbing wound exudate,include hydrocolloids, collagen-based materials, hyaluronic acid basedmaterials, dextrinomers, dextrinomer/cadexomer and oxidised regeneratedcellulose.

Dressings are typically provided in the form of a solid, dry non-wovensheet (or ‘felt’), a freeze-dried sheet, a ribbon or a rope, and areparticularly suitable for treating highly-exuding wounds.

Exemplary commercially available alginate-based materials includeSuprasorb® (Sammons Preston, USA) and Kaltostat® (ConvaTec, UK).

The dressing may comprise or consist of a sheet hydrogel. Such woundcare materials are particularly suitable for treating non-exudingwounds. Suitable sheet hydrogels may comprise one or morehydrogel-forming polymers selected from the group consisting ofsynthetic polymers, such as polyurethanes, polyvinylalcohol,polyvinylpyrrolidone, polyacrylic acid, polyethylene glycol, poloxamerblock copolymers and the like; semi-synthetic polymers, such ascellulose ethers, including hydroxyethylcellulose,hydroxypropylcellulose, methylcellulose, methylhydroxypropylcelluloseand ethylhydroxyethylcellulose, and the like; natural gums, such asacacia, carragenan, chitosan, pectin, starch, xanthan gum and the like;and alginates.

Exemplary sheet hydrogels available commercially include Elastogel®(Southwest Technologies Inc., USA) and Suprasorb® G (Sammons Preston,USA).

As a further alternative, the dressing may comprise or consist of ahydrofibre. Wound care products comprising such dressings are typicallyprovided in the form of a dry, non-woven sheet, freeze-dried sheet, or aribbon or rope, and are particularly suitable for use withlight-to-heavy exuding wounds or wounds with both dry and wet regions.

Suitable hydrofibres may comprise or consist of carbomethylcellulose,and include Aquacel® and Versiva® (both ConvaTec, UK) and Solveline® N(Activa Healthcare, UK).

As a further alternative, the dressing may comprise or consist of apolyurethane foam, such as the Allevyn® range of products (Smith &Nephew, UK), Mepilex®, (Mölnlycke, Sweden) and Tegaderm® (3M, UK).

Prior to use, the pharmaceutical formulation and the dressing should besterile and packaged in a microorganism-impermeable container. Sterilitymay be achieved using techniques well known in the art, such as asepticmanufacturing and/or final (i.e. post-production) sterilisation byirradiation.

As stated above, the dressing material should be inert, and thus notinteract in any way with LL-37, for example to degrade it chemically orphysically.

Dressings may thus also or alternatively comprise synthetic polymers,starches and/or polysaccharides. For example, the dressing may comprisean aqueous polymer matrix, a cellulose derivative, an acrylatecopolymer, a gum, a polysaccharide and/or a polylactic acid polymer.Such materials may be water-soluble and/or may be perforated.

In the method/uses of the invention, the formulation comprising LL-37 isapplied directly to the ulcer at the correct dosage as described aboveand the dressing thereafter applied to cover the treated wound area.

Optionally, a secondary conventional dressing may be applied over thetop of the first dressing. Furthermore, in some cases, a permeableanti-adherence dressing may be applied somewhere between the ulcer andthe dressing.

After application of the dressing, an appropriate compression means mayadvantageously be applied, including ductile, soft and absorbentcushions, such as Cellona® (Activa, UK) or Soffban® (Smith & Nephew,UK), compression bandages, such as Comprilan® (Smith & Nephew), and/ormedical stockings, such as ZipZoc® (Smith & Nephew).

The skilled person will appreciate that, prior to carrying out themethod/uses of the invention, it may be necessary to remove dead,damaged or infected tissue (debridement), treat any infected tissueusing an appropriate medical treatment (antiseptics), clean the woundarea using an appropriate cleaning agent (e.g. water), and dry it usinge.g. gauze. Curettage may also be conducted beforehand.

Moreover, and advantageously, the formulation and/or dressing may alsobe capable of preventing, abolishing, reducing or otherwise diminishingmicrobial growth in a wound environment. Thus, the formulation that isemployed in the method/uses of the invention may further comprise anantimicrobial substance, for example a polypeptide selected from thegroup consisting of defensins, gramicidin S, magainin, cecropin,histatin, hyphancin, cinnamycin, burforin 1, parasin 1, protamines, andfragments, variants and fusion thereof which retain, at least in part,the antimicrobial activity of the parent protein; as well as silver,silver sulfadiazine, polyhexinide, iodine or metronidazole.

It will be appreciated that, in the method/uses of the invention, theformulation, dressings and/or compression means should be replaced onthe wound at regular intervals, to aid the healing process and toprevent infection.

Whenever the word “about” is employed herein, particularly in thecontext of in relation to amounts, including doses of LL-37 andconcentrations in formulations, timings, viscosities, dosing factors,degrees of hydrolysis, molecular weights, etc., it will be appreciatedthat such variables are approximate and as such may vary by ±10%, forexample ±5% and preferably ±2% (e.g. ±1%) from the numbers specifiedherein.

The method/uses of the invention provide a wound care means that is easyand inexpensive to manufacture, and which enable the effective treatmentof chronic wounds, including VLUs, DFUs and the like.

The method/uses of the invention also have the advantage that theyemploy using established pharmaceutical processing methods and employmaterials that are approved for use in foods or pharmaceuticals, medicaldevice products, or of like regulatory status.

The method/uses of the invention may also have the advantage that theymay be more efficacious than, produce fewer side effects than, possess abetter patient acceptability than, have a better pharmacokinetic profilethan, and/or have other useful pharmacological, physical, or chemicalproperties over, similar methods known in the prior art, whether for thetreatment of chronic wounds, such as VLUs, DFUs, or otherwise.

The invention is illustrated by way of the following example, withreference to the attached FIG. 1, which shows estimates from a “brokenstick” analysis of wound healing rates by applying formulationscomprising different concentrations of LL-37 to HTH VLUs, in which thesolid line represents the placebo group, the large dashed linerepresents 3.2 mg/mL group, the smaller dashed line represents 1.6 mg/mLgroup, and the dotted line represents 0.5 mg/mL group.

EXAMPLE Double-Blind, Randomised, Placebo-Controlled, Safety andPilot-Dose Response Study of LL-37 in HTH VLUs

A double-blind, randomised, placebo-controlled, Phase I/II safety andpilot-dose response study was conducted on human subjects. The firstsubject was enrolled in August 2012 and the last subject completed inApril 2013.

The study was designed, conducted and reported in accordance with theInternational Conference on Harmonisation (ICH) Harmonised TripartiteGuidelines for Good Clinical Practice (GCP), with applicable localregulations and ethical principles laid down in the Declaration ofHelsinki and was conducted across three centres in Sweden.

Selection Criteria

Study subjects comprised either male subjects of at least 18 years ofage, or post-menopausal or clinically sterile women, with VLUs orcombined venous-arterial ulcers with a predominant venous component.

Exclusion criteria included if subjects had known predominant aetiologyother than VLU within the target ulcer, and malignant disease (excludingbasal cell carcinoma), unless in remission for 5 years or more.

After obtaining necessary regulatory (Swedish Medicinal Products Agency(MPA) and ethical (regional Ethics Committee) approvals, and writteninformed consent, a total of 56 subjects attended a screening visitfollowed by a 3-week open-label run-in period.

Target wounds with an area greater than 30 cm² and less than 2 cm² wereexcluded initially. However, in order to increase the inclusion rate anddecrease the total study time, criteria were amended after randomisationof 21 subjects, to exclude areas greater than 40 cm² and less than 1cm². If an eligible subject had more than one wound satisfying the entrycriteria, the investigator identified the wound with the mostappropriate size, shape and localisation as target wound. Only one woundper subject was treated in the study.

During the 3-week run-in period, subjects received, every third day (±1)and not more than two times per week, six applications (in total) of aplacebo preparation (see below) in combination with standard compressionbandaging (as defined by the Swedish MPA), comprising elastic bandagingand appropriate dressing to control wound exudation.

If the ulcerative area decrease was greater than between 7% and 13%(depending on size) per week relative to the start of the run-in period,the ulcer was not defined as HTH for the purposes of this study. In suchcases, subjects were excluded from the study and the outcome wasrecorded as screening failure.

After a run-in period, 34 eligible subjects underwent baselineassessments before randomisation to receive active treatment (one ofthree treatment strengths) or placebo.

Formulations and Treatment

Following the above run-in period, a 4-week treatment period wasinitiated, including 8 visits, an end-of-treatment visit 3 days (±1 day)after the last treatment, and a follow-up visit after an additional 4weeks (±1 to 7 days).

Subjects were randomised into four treatment groups to receive LL-37(one of three alternative strengths), or placebo.

Wound bed preparation prior to treatment included debridement ofnon-viable tissue, treatment of clinical infection and selection of themost appropriate dressing (see below). The wound was cleaned using tapwater, gently dried with a gauze pad and, if necessary, curettage wasapplied prior to application of the trial product or placebo.

0.7 mL of a sterile concentrate of LL-37 in water at concentrations of2.5, 8 or 16 mg of LL-37 per mL was provided in sealed glass vials (APL,Umeå, Sweden), which were stored prior to the trial at between 2 and 8°C. Immediately before application to the wound, this was diluted with 5mL of a viscous diluent solution comprising 13.1% (w/w) of polyvinylalcohol in acetate buffered saline (APL AB) to give final concentrationsof either 0.5, 1.6 or 3.2 mg/mL, respectively. Placebo comprised exactlythe same solution, but without LL-37 present.

The diluted viscous liquid solution of active treatment or placebo wasapplied topically at an amount of 25 μL/cm² with a 1 mL graded syringeat the centre of the cleansed and dried ulcer, and distributed evenlyover the entire ulcer area using the rounded head of the syringe plungerthat had been used in mixing the study drug.

After a 5-minute wait (to prevent LL-37 or placebo from being absorbedby the dressing material), the above procedure was followed byapplication of a dressing and a compression bandage.

For moderate to heavy exudating wounds, highly absorbent dressings madeof polyurethane foam, such as Mepilex®, Allevyn® and Tegaderm®, wereused. Alternatively, the absorbent hydrofibre dressing Aquacel® wasused. For slight to moderate exudative wounds, non-adherent dressingswere used, with or without an adhesive edge. The dressings used wereSolvaline® N or the combined polyurethane/hydrofibre dressing Versiva®.For dry wounds, Aquacel® hydrofibre dressing was used.

In combination with these other dressings, gauze pads were used tooffload sensitive parts during compression. A 100% viscose, ductile,soft and absorbent cushion was used, including Cellona® or Soffban®.Comprilan® was the most commonly used compression bandage used in thestudy. Two bandages (8 and 10 cm wide) were tied with approximately 50%stretch from the base of the toes in a spiral to just beneath the knee.Alternatively, ZipZoc® medical stocking plus Coplus® or a similarself-adhesive bandage or Profore Lite® in combination with some of theabove mentioned wound dressings were allowed. A long-stretch bandage ofDauer-type could also be used. Because of the high resting pressure withthis type of bandage, it was temporarily removed during the night orwhen the subject was in a resting position for a long period.

Additional treatment included specific topical creams for the treatmentof skin problems. Zinc paste or Cavilon® were used on per-ulcer skin toprotect the skin near the wound edge from maceration. Hydrocortisonecream or ointment (topical steroid group I or hydrocortisone-imidazolecream) was used for peri-ulcer eczema. If the skin was dry and scaly,Canoderm or Propyderm cream was allowed.

The above procedure was repeated every third day (±1) over a 4 weektreatment period, not more than two times per week, representing a totalof eight applications.

Subsequent to treatment, subjects were followed up for a period of 4weeks (±1 to 7 days).

The study assessed the rate of wound healing, pain at wound site both atthe time of dressing and over the previous 24 hours using VisualAnalogue Scale (VAS). Any incidence of complete healing was recordedeven though statistically significant results were not expected based onthe short duration of the trial and the small sample size. Wound sizemeasurements were performed repeatedly over the study period to indicatethe extent of wound healing. At every other visit, wound area wasmeasured using the Visitrak™ planimetry device, pain at the wound siteand wound characteristics (odour, slough, granulation and necrotictissue) were recorded. The wound condition was to be described usingphotographs of the wound.

Adverse events (AEs) and local tolerability were assessed at all visits.Subjects were advised to report any AEs and were given an emergency cardwith a phone number to call in case of any significant AEs. Blood wascollected for laboratory safety assessment and investigation ofdevelopment of antibodies against LL-37. Changes in laboratory valuesand vital signs were assessed, and analysis of auto-antibody formationagainst LL-37 was performed.

Safety endpoints included:

-   -   incidence of severe local reactions in wound and adjacent skin        as exemplified by clinical signs of inflammation (oedema,        redness, odour and raised temperature) or skin irritation        (scaling, redness, papules, vesicles, pustules); any local        reaction was recorded on a graded scale (0 to 3);    -   incidence of greater than 30% increase in wound area relative to        baseline (randomisation visit);    -   change in laboratory values and vital signs from baseline;    -   overall incidence of AEs.

Efficacy endpoints included:

-   -   wound healing rate within the study period;    -   a number of ulcers attaining a greater than 30% area reduction        from baseline at randomization; and    -   changes in local pain using VAS score (0 to 10)    -   changes in wound characteristics: (scores for slough, exudation,        granulation tissue, necrosis) from baseline (scale 0 to 3).

Analyses

Statistical analyses were carried out using SAS® software (version 9.2or higher; SAS Institute, Cary, N.C., US).

The wound area was modelled over time for each subject to estimate thehealing rate over the treatment period and the relationship betweenhealing rate and dose was investigated.

Estimates of healing rate constants (i.e. the change in wound area ormathematically transformed wound area/day) were derived with 95%confidence intervals (CIs) for each of the four treatment groups. Anexponential decay model was fitted to the data with two parametersrepresenting the initial wound area and the rate of healing. The modelwas of the form:

Y=α×e ^(βt)

where α denotes the initial wound area (i.e. at randomisation visit), βdenotes the reduction in size expressed as healing rate per day, Ydenotes the wound area and t denotes the time in days from therandomisation visit.

Additional analyses incorporating all run-in data was conducted toassess how the application of study treatments affected the healingprocess. This was assessed using a “broken-stick” approach,approximating pre- and post-randomisation data by a linear response overtime after suitable data transformation (e.g. log, square root).

If appropriate, pain scores were analysed using a repeated measuresanalysis to assess the overall difference between treatments over time.The model included terms for treatment, and time and the interactionbetween the two.

The incidence of any AEs was estimated using the percentage of subjectsin each treatment group who experienced at least one AE, with associated95% CIs. CI was calculated using the method detailed in Altman et al,Statistics with Confidence, BMJ Publication Group (2000), which wasappropriate for small samples, or low incidences. The relationshipbetween incidence of AEs and dose was investigated if appropriate. Theoverall incidence of grade 3 (severe) local reactions was presented forthe LL-37 treated subjects with 95% CIs. Laboratory data were to beshown in plots of post-treatment values versus baseline, with referenceranges.

Results

A total of 31 subjects received the full course of eight treatmentapplications. Of the remaining three subjects, two (in 0.5 mg/mLtreatment group) received seven applications and one subject (in placeboarm) received three applications.

The exponential decay model fitted to the wound area provided a good fitto the data.

The healing rate constant for the 0.5 mg/mL dose of LL-37 (0.039)differed significantly from that for placebo (0.007, p=0.003)representing a faster reduction in wound area (i.e. improved healing). Anon-linear mixed effect model was used and the significance of thedifference between the parameters was calculated by taking the estimateof the difference and dividing by its standard error, and comparing theresult against a t-distribution.

The estimated healing rate constant for the 1.6 mg/mL dose of LL-37,0.019, also represented a faster reduction in wound area compared toplacebo, although was not statistically significant at the 5% level(p=0.09). There was no statistically significant difference between thehighest dose of LL-37 (healing rate constant 0.004) group and theplacebo group. The low and intermediate dose groups were statisticallyindistinguishable. The estimated healing rate constants, with a measureof mean reduction in wound area, for each treatment are shown in Table 1below.

TABLE 1 Estimates of wound healing rates Estimated Estimated healingEstimated difference initial area rate constant¹ relative to control(cm²) Mean (cm²/day) Mean (cm²/day) Mean p- Treatment (95% C.I.) (95%C.I.) (95% C.I.) value Placebo 9.222 (6.133 0.007 (−0.001 (n = 9) to12.312) to 0.014) LL-37 0.5 5.036 (1.742 0.039 (0.020 0.032 (0.012 0.003mg/mL to 8.329) to 0.058) to 0.053) (n = 8) LL-37 1.6 7.498 (4.583 0.019(0.007 0.012 (−0.002 0.088 mg/mL to 10.414) to 0.031) to 0.026) (n = 9)LL-37 3.2 5.872 (2.573 0.004 (−0.007 −0.003 (−0.016 0.656 mg/mL to9.171) to 0.015) to 0.010) n = 8) ¹Where the lower 95% confidence limitis negative this represents a positive healing rate constant over timesince the model fitted assumes a negative constant (i.e. decrease inwound area).

Overall, the highest reduction in wound area was noted for 1.6 mg/mLdose of LL-37 across all visits. The mean reduction in wound area for1.6 mg/mL dose of LL-37 was 5.29 cm² from randomization to the end ofthe study (see Table 2 below).

TABLE 2 Wound Area (cm²) at Each Visit Mean ± S.D for the visits LL-37LL-37 LL-37 Placebo 0.5 mg/mL 1.6 mg/mL 3.2 mg/mL (N = 9) (N = 8) (N =9) (N = 8) Visit 1 9.72 ± 8.475 4.59 ± 2.024 7.84 ± 4.789 8.41 ± 6.450(Screening) Visit 3 10.25 ± 9.875  4.39 ± 1.995 8.14 ± 4.997 7.83 ±6.163 Visit 6 9.54 ± 8.246 5.11 ± 1.732 7.69 ± 4.072 8.50 ± 6.919(Random- isation) Visit 8 8.60 ± 8.171 3.89 ± 1.329 5.83 ± 2.156 7.95 ±6.270 Visit 10 6.63 ± 5.569 3.18 ± 1.549 5.34 ± 1.943 7.03 ± 5.063 Visit12 6.50 ± 7.777 2.26 ± 1.061 4.59 ± 2.266 5.93 ± 5.317 Visit 14 6.16 ±6.850 1.93 ± 1.262 3.86 ± 3.029 5.89 ± 4.784 Visit 15 6.52 ± 7.683 1.98± 1.276 3.34 ± 3.166 5.85 ± 4.648 (End of Treatment) Visit 16 5.76 ±7.574 1.33 ± 1.209 2.40 ± 3.269 6.19 ± 4.770 (End of Study) SD: Standarddeviation; end of study refers to the follow-up visit performedapproximately 4 weeks after the last treatment.

The healing rate constant for the exponential decay model exhibited a6-fold difference for the lowest dose (0.5 mg/mL) group, and a 3-folddifference for the intermediate dose (1.6 mg/mL) group versus theplacebo group (p=0.003 for 0.5 mg/mL and p=0.088 for 1.6 mg/mL). Theseformulations thus gave rise to a faster reduction in wound area, andthus an improved healing response. However, there was no differencebetween the estimated healing rates in the highest LL-37 dose (3.2mg/mL) group and the placebo group.

In order to incorporate the data from the run-in and to assess if theaddition of treatment affected the rate of healing, a broken stickanalysis was conducted and was consistent with the previous analyses. Asshown in FIG. 1, both the lower two doses of LL-37 had greater reductionin wound area after randomisation than during the run-in, indicatingstatistically significant improved healing compared to pre-treatmentvalues (p<0.001 and p=0.011 for 0.5 mg/mL and 1.6 mg/mL respectively).No significant difference between the post-randomisation rate and therun-in was seen for either the highest dose or placebo. The rate ofchange during the run-in was very close to zero, as would be expectedsince responders were excluded from the study.

CONCLUSIONS

A marked and significant improvement in wound healing was observed inresponse to administration of the two lower doses of LL-37 (0.5 and 1.6mg/mL). The results from this study suggest that doses of 0.5 (whichequates to 13 μg of LL-37 per cm² of wound area), and 1.6 mg/mL (whichequates to 40 μg of LL-37 per cm² of wound area) every third day resultsin significant stimulation of wound healing. Alternatively, a lessfrequent application of a higher dose may be appropriate.

1. A method of treating a chronic ulcer, which method comprises (a)topically applying to said chronic ulcer a pharmaceutical formulationcomprising LL-37, or a pharmaceutically-acceptable salt thereof, and oneor more pharmaceutically-acceptable diluent or carrier system (a) ;followed by (b) application of a dressing; wherein said application ofsaid formulation provides for a dose of LL-37 at the wound site that isbelow about 80 μg of LL-37 applied per cm² of wound area.
 2. A method asclaimed in claim 1, wherein the dose of LL-37 is below a value that isbetween about 40 μg/cm² and about 80 μg/cm².
 3. A method as claimed inclaim 1, wherein the dose of LL-37 is between about 1 μg/cm² and about70 μg/cm².
 4. A method as claimed in claim 1, wherein the dose isbetween about 5 μg/cm² and about 60 μg/cm².
 5. A method as claimed inclaim 1, wherein the dose is below about 26.7 μg/cm² per day oftreatment.
 6. A method as claimed in claim 5, wherein the dose of LL-37is below a value that is between about 13.3 μg/cm² and about 26.7 μg/cm²per day of treatment.
 7. A method as claimed in claim 5, wherein thedose of LL-37 is between about 0.3 μg/cm² and about 23.3 μg/cm² per dayof treatment.
 8. A method as claimed in claim 5, wherein the dose isbetween about 1.6 μg/cm² and about 20 μg/cm² per day of treatment.
 9. Amethod as claimed in claim 1, wherein the formulation is a viscousliquid or a semi-solid.
 10. A method as claimed in claim 9, wherein theviscosity of the formulation in the range of about 2 and about 25 Pa·s.11. A method as claimed in claim 1, wherein the chronic ulcer is avenous leg ulcer.
 12. A method as claimed in claim 1, wherein thechronic ulcer is a diabetic foot ulcer.
 13. A method as claimed in claim1, which comprises an aqueous solution of LL-37.
 14. A method as claimedin claim 1, which is in the form of a suspension, an emulsion, a cream,a gel or a lotion.
 15. A method as claimed in claim 1, which comprisespolyvinyl alcohol.
 16. (canceled)
 17. (canceled)